Cofactor-free detection of phosphatidylserine with cyclic peptides mimicking lactadherin

Article abstract of DOI:10.1021/ja205911n

Cyclic peptides (cLacs) are designed to mimic the natural phosphatidylserine (PS) binding protein lactadherin. Unlike annexin V or its small molecule mimics, the cLac peptides selectively target PS-presenting membranes with no need for metal cofactors. We further show that a fluorophore-labeled cLac effectively stains early apoptotic cells. The small size and facile conjugation with a variety of imaging tracers make the cLac design promising for imaging cell death in vitro as well as in living organisms.

Full text of DOI:10.1021/ja205911n

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pubs.acs.org/JACS
Cofactor-Free Detection of Phosphatidylserine with Cyclic Peptides
Mimicking Lactadherin
Hong Zheng, Fang Wang, Qin Wang, and Jianmin Gao*
Department of Chemistry, Merkert Chemistry Center, Boston College, 2609 Beacon Street, Chestnut Hill, Massachusetts 02467,
United States
S Supporting Information
b
we grafted the key residues of the PS-targeting protein lactadher-
ABSTRACT: Cyclic peptides (cLacs) are designed to
mimic the natural phosphatidylserine (PS) binding protein
lactadherin. Unlike annexin V or its small molecule mimics,
the cLac peptides selectively target PS-presenting mem-
branes with no need for metal cofactors. We further show
that a fluorophore-labeled cLac effectively stains early
apoptotic cells. The small size and facile conjugation with
a variety of imaging tracers make the cLac design promising
for imaging cell death in vitro as well as in living organisms.
in into the scaffold of a cyclic peptide. In contrast to the
organozinc complexes, the cyclic lactadherin mimics (cLacs)
do not require any cofactors for PS recognition. We show that a
fluorophore-labeled cLac selectively stains apoptotic cells by
targeting PS on cell surfaces.
Lactadherin is a 47-kDa glycoprotein originally found in the
milk secreted by mammary epithelial cells.19 One of its key
functionsof lactadherin is to mediatethephagocytosis of apoptotic
cells by recognizing surface-exposed PS.20 Lactadherin stereose-
lectively binds to PS with a nanomolar affinity and without the
need for cofactors.21 Fluorescently labeled lactadherin isbeginning
tobe used for PSdetectionon stored platelets6 and cultured cancer
cells.22,23 The PS binding potential of lactadherin is localized to its
C2domain (Lact-C2),24 thestructureof whichwas recently solved
through X-ray crystallography.25,26 The lact-C2 structure displays
a beta-barrel core and three loops projecting out for membrane
insertion (Figure S1, Supporting Information (SI)). While the
structure of the Lact-C2-PS complex remains elusive, the muta-
genesis data as well as computational modeling suggest two
potential binding sites (Surfaces C and D, as named by Shao
et al.26) for the PS headgroup.26 We focused on Surface D because
computational docking experiments gave a more favorable binding
free energy for the PS headgroup than PC (SI). Further structure
examination revealed a number of residues critical for PS recogni-
tion (Figure 1): Phe81 and Trp26 belong to the group of
hydrophobic residues that insert into the membrane; the hydro-
philic residues (Asp80, His83, Gln95, and Arg148) engage in
specific polar interactions with the PS headgroup that afford the
lipid selectivity. The PS binding pocket looks like a bowl with the
key residues displayed on the rim. Based on this observation, we
hypothesize that the circular arrangement of these key residues can
be recreated by the scaffold of a cyclic peptide.
Our first design (cLac-1, Figure 1) positions a Trp and a Phe
next to each other to construct a membrane insertion motif.
Three of the four polar residues (Asp80, His83, and Gln85)
involved in PS binding are close in the Lact-C2’s primary
sequence. One exception is Arg148, which projects back into
the vicinity of the other key residues in the folded protein. For the
design of cLac-1, we linked Arg148 to the rest of the molecule by
using two spacers: a dipeptide (-Gly-Gly-, Linker I) bridges
Gln85 and Arg148, and a tripeptide (-Gly-Gly-Gly-, Linker II)
links Arg148 and Trp26. Molecular modeling of cLac-1 shows
that the two linkers will be sufficiently long and flexible to allow
he lipid composition and distribution of cell membranes play
T
an important role in regulating the physiology of the cell. For
example, many signaling proteins can be recruited to the
membrane by clusters of negatively charged lipids.1À3 Not
surprisingly, live mammalian cells actively maintain an asym-
metric lipid distribution on their plasma membrane, with the
most abundant anionic lipid phosphatidylserine (PS) completely
confined to the cytosolic leaflet.4 PS externalization has been
observed for cells under abnormal development, including
apoptotic cells,5 activated platelets,6 and virus-infected cells.7
In fact, imaging PS exposure has been a benchmark approach to
detect apoptotic cells in biological research. Considering most
cancer therapeutics are designed to elicit apoptosis of cancer
cells, noninvasive imaging of PS may enable facile evaluation of
the efficacy of anticancer drugs.
Among the limited number of PS-targeting ligands,8 annexin V
has been most extensively investigated for detecting apoptotic
cells in vitro and in living organisms. This 36-kDa protein, labeled
with fluorochromes or radioactive nuclides, has been studied for
imaging cell death in animal models of human cancer,9 as well as
in humans.10 However, annexin V is less than ideal for PS imaging
due to its large size, high cost, and calcium dependence of PS
recognition. In addition, the moderate stability and slow clear-
ance of annexin V severely limit its application in living
organisms.11,12 Small molecules that specifically target PS may
circumvent the problems afflicting annexin V, and their devel-
opment has captured increasing attention.8 The elegant work
from the Smith group resulted in a class of bivalent organozinc
complexes that functionally mimic annexin V and preferentially
associate with negatively charged membranes.13À16 Recently,
screening of phage-displayed peptide libraries has also yielded
promising leads for PS recognition, although the molecular
mechanisms of PS specificity have not been described for these
peptides.17,18 Herein, we report a new class of rationally designed
small molecule receptors for PS. Taking a biomimetic approach,
Received: June 24, 2011
Published: September 07, 2011
r
2011 American Chemical Society
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Figure 2. PS-targeting cyclic peptides. (a) Structure of cLac-2 that incor-
porates biphenylalanine (Bip) as a membrane anchor; (b) cLac-2 (at 25 μM)
sensitizes dansyl emission (highlighted by the arrows) only with PC/PS
(20%) liposomes; (c) concentration profiles of cLac-2 binding to mem-
branes with varied composition; (d) SAR (structureÀactivity relationship)
study of cLac-2 to compare the peptide variants with different linker lengths;
all peptides are used at 25 μM concentration.
Figure 1. Structural modeling of Lact-C2 binding a short chain
(diacetyl) PS, leading to the design of cLac-1 that mimics the PS binding
pocket of lactadherin. The model is based on the Lact-C2 structure
PDB: 3BN6.
the native-like spatial arrangement of these residues for PS
recognition.
constant (Kd) of ∼8 μM. With cLac-2 at the saturating con-
centration (25 μM), the sensitized dansyl emission displays a
linear dependence on the amount of PS presented by the
membranes: vesicles containing 20% PS give close to 4-fold as
much dansyl fluorescence as that of 5% PS (Figure 2c). It is worth
noting that cLac-2 displays no net charge under experimental
conditions, suggesting its selective association with PS-present-
ing membranes is not merely driven by the electrostatic attrac-
tion to the negatively charged surfaces. This compares favorably
to the cationic peptide-based ligands17 and the organozinc
complexes,15 which target PS predominantly through Coulombic
interactions.
Further study of the structureÀactivity relationship shows that
the linear precursor of cLac-2 (Lac-linear) binds to neither type
of vesicle (Figure S3, SI), indicating that the circular arrangement
of the key residues is necessary for the PS-dependent membrane
association of the peptide. Our initial optimization of cLac-2
focused on the oligoglycine linkers. The number of Gly residues
within Linker II was varied from 2 to 5. The Trp-to-dansyl FRET
results (Figure 2d) show that cLac-22G (diglycine as linker II)
elicits minimal dansyl emission, presumably because the linker is
too short to allow the proper ring geometry for PS binding.
Lengthening the linker to the tri- and tetraglycine moieties
results in cLac-2 and cLac-24G respectively, which display a
continued increase in dansyl emission, indicating an improved
affinity of cLac for the membrane. Further extension to the
pentaglycine linker affords cLac-25G that gave a reduced dansyl
emission, presumably because the excessive flexibility of the
linker causes an overwhelming entropic penalty toward mem-
brane binding. These data indicate that the cLac-PS binding
requires the cooperative action of multiple residues of the cLac
peptide, analogous to the lipid binding mode of lactadherin, the
protein that nature evolved for PS recognition. Interestingly, the
peptide variants with varied lengths of Linker I (cLac-13G, cLac-
2, and cLac-33G) exhibit a much less significant difference in the
The cyclic peptide was synthesized by using Fmoc/tBu
chemistry (Scheme S1, SI). The main chain protected (with
allyl) Asp was attached to Wang resin through its side chain
carboxyl group. Standard solid phase peptide synthesis af-
forded the linear precursor with a deprotected N-terminus.
Cyclization was accomplished on resin with HBTU-mediated
amide bond formation after removal of the allyl protecting
group on Asp. The peptide-membrane binding was evaluated
by using a fluorescence assay: large unilamellar vesicles
(∼100 nm in diameter) were prepared with 1-palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholin (POPC) and varied per-
centages of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-ser-
ine (POPS). All vesicles contained 5% dansyl-labeled
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
(POPE) to serve as a reporting group. The peptide-vesicle
binding was monitored by the fluorescence resonance energy
transfer (FRET) from Trp to the dansyl group. Our starting
design cLac-1 displays no binding to lipid vesicles regardless of
lipid composition (Figure S2, SI). Presumably, the peptide is
too hydrophilic to partition into the membranes. This is
perhaps not surprising given that additional hydrophobic
residues (Leu28 and Phe31) are necessary for Lact-C2 to
insert into membranes.26
A more hydrophobic variant of cLac (cLac-2, Figure 2a) was
synthesized, which incorporates a biphenylalanine (Bip) to
replace the Phe residue. The side chain of Bip has a cLogP value
of 4.5, significantly greater than that of Phe (cLogP: 2.6). The
FRET experiment shows no binding of cLac-2 to the PC-alone
liposomes. However, mixing cLac-2 with the PC/PS vesicles
clearly elicits a sensitized dansyl emission at 520 nm (Figure 2b).
Furthermore, the sensitized dansyl emission becomes stronger
with increasing concentrations of cLac-2. The titration results
display a saturation profile, yielding an apparent dissociation
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Figure 4. Flow cytometry results illustrating the effectiveness of the
cLac peptide in labeling apoptotic cells. Jurkat cells treated with (A, C)
and without (B, D) camptothecin were analyzed with cLac-2-Fl (A, B)
and FITC labeled Annexin V (C, D) respectively. For each graph, the cell
population is plotted against the fluorescence intensity (in log scale) of
the cells. The percentage of cell population and the mean fluorescence
intensity are labeled for each peak.
Figure 3. Microscopy analysis of cLac-2-Fl in staining apoptotic cells.
(A) Phase contrast; (B) FITC channel; (C) PI channel; (D) overlay of B
and C. Little staining of the nonapoptotic cells was observed for cLac-2-Fl.
peptides’ binding to membranes, possibly because the variation
of Linker I can be effectively attenuated by the flexible Linker II
(triglycine) on the other side of Arg148.
staining and PI fluorescence, suggesting cLac largely detects
early apoptotic cells. A similar phenomenon was observed for
annexin V (Figure S5, SI). Since these early apoptotic cells
hold their membrane integrity (negative for PI staining), we
believe cLac2-Fl stained the cells by binding to their outer
surfaces, presumably to PS.
In order to evaluate the potential of cLac as an imaging reagent
for apoptotic cells, we functionalized cLac-2 with a fluorescein
label through the bioorthogonal click chemistry.27,28 A propargyl
glycine (Pra) was introduced to replace the Ile residue within the
cLac-2 sequence. Based on the Lact-C2 structure (Figure S1, SI),
the Ile side chain projects away from the proteinÀPS interface.
Therefore we expect the Ile-to-Pra mutation to introduce mini-
mal perturbation to the peptideÀmembrane association. Indeed,
when the Pra derivative (cLac-2-Pra) was measured against
liposomes with and without PS, membrane selectivity essentially
identical to the parent peptide (Figure S4, SI) was observed.
A fluorescein moiety was conjugated onto cLac-2-Pra to give cLac-
2-Fl via the bioorthogonal click chemistry (SI), in which a flexible
PEG linker bridges the fluorophore and the cyclic peptide. This
design separates the fluorophore and the peptide so the specific
binding of cLac and the membrane is minimally perturbed.
The fluorescent cLac was tested for its potential to stain
apoptotic cells. Cultured HeLa cells were treated with the potent
apoptogen staurosporine (STS) to trigger apoptosis. The experi-
mental conditions were optimized with the FITC-labeled annex-
in V and propidium iodine (PI, a membrane impermeable DNA
intercalator). In our hands, treatment with 1 μM STS for 3 h gave
a large population of early apoptotic cells, which were stained
with annexin V, but not PI (Figure S5, SI). Under these
conditions, cLac-2-Fl (the green channel) readily stained STS
treated cells, while little fluorescence was observed for the
untreated cells (Figure 3). The lack of staining for the untreated
cells indicates that cLac-Fl does not gain entry into healthy cells
by diffusion or by endocytosis, as expected for most peptide-
based agents. As a negative control, we tested the FITC-labeled
Lac-linear, which gave little staining of the apoptotic cells under
the same experimental conditions (Figure S6, SI). Collectively
these results indicate cLac-2-Fl recognizes apoptotic cells
through a specific mechanism. The merged microscopic image
(channel D, Figure 3) exhibits minimal overlay between cLac
To test the generality of the cLac peptide, we further
examined its potential in imaging Jurkat cells with both
microscopy and flow cytometry. Jurkat cells were treated with
and without camptothecin (CPT) and then stained with cLac-
2-Fl and the FITC labeled annexin V, respectively. Analogous
to the HeLa cells, cLac-2-Fl readily differentiated the drug-
treated versus untreated Jurkat cells (bottom panel, Figure 3).
The cLac staining of Jurkat cells was quantitatively evaluated
via flow cytometry analysis (Figure 4). Similar to annexin V,
which stains ∼16% of the CPT-treated cells, cLac-2-Fl labels
∼18% of the cell population of the identical sample. Presum-
ably cLac-2 and annexin stain the same subpopulation of the
cells that is at the early stages of apoptosis. Lower fluorescence
intensity was observed for the apoptotic cells treated with
cLac-2-Fl than annexin V. This is partly because annexin V
carries multiple copies of fluorophores (conjugated onto
lysine side chains), while cLac-2 is labeled with a single dye.
The relatively low PS-binding affinity of the cLac peptide may
also contribute to the low fluorescence intensity observed for
the apoptotic cell population.
In summary, we have developed a new class of cyclic peptides
that successfully mimic the function of the natural PS-binding
protein lactadherin. These cyclic lactadherin mimics (cLacs)
selectively associate with PS-presenting membranes with low
micromolar affinity. Furthermore, we demonstrate with two
different cancer cell lines that a fluorescently labeled cLac
effectively detects cells at the early stages of apoptosis. Impor-
tantly, the cLac-PS recognition does not require any metal
cofactors and compares favorably to annexin V, whose PS-
binding affinity is highly sensitive to calcium concentrations
(see ref 29 and also Figure S8, SI). While the PS-binding affinity
and the brightness of fluorescent labels remain to be optimized,
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the cLac design benefits from its small size, ease of labeling, and
cofactor-free PS recognition, which is highly promising for
imaging apoptosis in cell cultures as well as in living organisms.
In addition, the biomimetic approach that we used for cLac
should be extendable to the development of small molecule
receptors for other lipid molecules. Research toward these
directions is currently ongoing.
(19) Hvarregaard, J.; Andersen, M. H.; Berglund, L.; Rasmussen,
J. T.; Petersen, T. E. Eur. J. Biochem. 1996, 240, 628.
(20) Hanayama, R.; Tanaka, M.; Miwa, K.; Shinohara, A.; Iwamatsu,
A.; Nagata, S. Nature 2002, 417, 182.
(21) Shi, J.; Heegaard, C. W.; Rasmussen, J. T.; Gilbert, G. E.
Biochim. Biophys. Acta 2004, 1667, 82.
(22) Waehrens, L. N.; Heegaard, C. W.; Gilbert, G. E.; Rasmussen,
J. T. J. Histochem. Cytochem. 2009, 57, 907.
(23) Shi, J.; Shi, Y.; Waehrens, L. N.; Rasmussen, J. T.; Heegaard,
C. W.; Gilbert, G. E. Cytometry A 2006, 69, 1193.
(24) Andersen, M. H.; Graversen, H.; Fedosov, S. N.; Petersen,
T. E.; Rasmussen, J. T. Biochemistry 2000, 39, 6200.
(25) Lin, L.; Huai, Q.; Huang, M.; Furie, B.; Furie, B. C. J. Mol. Biol.
2007, 371, 717.
(26) Shao, C.; Novakovic, V. A.; Head, J. F.; Seaton, B. A.; Gilbert,
G. E. J. Biol. Chem. 2008, 283, 7230.
(27) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004.
(28) Best, M. D. Biochemistry 2009, 48, 6571.
’ ASSOCIATED CONTENT
S
Supporting Information. Supporting Information avail-
b
able on peptide synthesis and characterization, fluorescence
labeling and imaging protocols, and results of the control
experiments. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(29) Tait, J. F.; Gilson, D. F.; Smith, C. Anal. Biochem. 2004,
329, 112.
Corresponding Author
Jianmin.gao@bc.edu
’ ACKNOWLEDGMENT
This work is supported by the Research Start-up Fund from
Boston College. We thank Prof. Eranthie Weerapana for her help
in the cell culture experiments. We also thank Dr. Josh Rosenberg
for his assistance on fluorescence imaging of cells and Dr. Patrick
Autissier for his help on the flow cytometry measurements.
’ REFERENCES
(1) Lemmon, M. A. Nat. Rev. Mol. Cell. Biol. 2008, 9, 99.
(2) Kutateladze, T. G. Nat. Chem. Biol. 2010, 6, 507.
(3) Hurley, J. H. Biochim. Biophys. Acta 2006, 1761, 805.
(4) Balasubramanian, K.; Schroit, A. J. Annu. Rev. Physiol. 2003,
65, 701.
(5) Schlegel, R. A.; Williamson, P. Cell Death Differ. 2001, 8, 551.
(6) Hou, J.; Fu, Y.; Zhou, J.; Li, W.; Xie, R.; Cao, F.; Gilbert, G. E.;
Shi, J. Vox Sang 2011, 100, 187.
(7) Soares, M. M.; King, S. W.; Thorpe, P. E. Nat. Med. 2008,
14, 1357.
(8) Hanshaw, R. G.; Smith, B. D. Bioorg. Med. Chem. 2005, 13, 5035.
(9) Ntziachristos, V.; Schellenberger, E. A.; Ripoll, J.; Yessayan, D.;
Graves, E.; Bogdanov, A., Jr.; Josephson, L.; Weissleder, R. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 12294.
(10) Belhocine, T.; Steinmetz, N.; Hustinx, R.; Bartsch, P.; Jerusalem,
G.; Seidel, L.; Rigo, P.; Green, A. Clin. Cancer Res. 2002, 8, 2766.
(11) Vermeersch, H.; Loose, D.; Lahorte, C.; Mervillie, K.; Dierckx,
R.; Steinmetz, N.; Vanderheyden, J. L.; Cuvelier, C.; Slegers, G.; Van de
Wiele, C. Nucl. Med. Commun. 2004, 25, 259.
(12) Edgington, L. E.; Berger, A. B.; Blum, G.; Albrow, V. E.; Paulick,
M. G.; Lineberry, N.; Bogyo, M. Nat. Med. 2009, 15, 967.
(13) Koulov, A. V.; Stucker, K. A.; Lakshmi, C.; Robinson, J. P.;
Smith, B. D. Cell Death Differ. 2003, 10, 1357.
(14) Smith, B. A.; Akers, W. J.; Leevy, W. M.; Lampkins, A. J.; Xiao,
S.; Wolter, W.; Suckow, M. A.; Achilefu, S.; Smith, B. D. J. Am. Chem. Soc.
2010, 132, 67.
(15) DiVittorio, K. M.; Johnson, J. R.; Johansson, E.; Reynolds, A. J.;
Jolliffe, K. A.; Smith, B. D. Org. Biomol. Chem. 2006, 4, 1966.
(16) Hanshaw, R. G.; Lakshmi, C.; Lambert, T. N.; Johnson, J. R.;
Smith, B. D. ChemBioChem 2005, 6, 2214.
(17) Burtea, C.; Laurent, S.; Lancelot, E.; Ballet, S.; Murariu, O.;
Rousseaux, O.; Port, M.; Vander Elst, L.; Corot, C.; Muller, R. N. Mol.
Pharmaceutics 2009, 6, 1903.
(18) Thapa, N.; Kim, S.; So, I. S.; Lee, B. H.; Kwon, I. C.; Choi, K.;
Kim, I. S. J. Cell. Mol. Med. 2008, 12, 1649.
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